Under resting conditions, intracellular calcium ion concentrations are very low. The rapid entry of calcium into cells is mediated by voltage-gated calcium channels, integral membrane proteins that respond to fast depolarizations of the membrane by transiently and reversibly opening a calcium-selective pore through the cellular membrane. This pore allows the rapid diffusion of calcium ions (the calcium current) from the extracellular medium, down their concentration gradient, to the intracellular space. Higher intracellular concentrations of calcium ions trigger a wide variety of cellular and physiological responses, including excitation-contraction coupling, hormone secretion and gene expression.
Since normal physiological functions are mediated by calcium channels, malfunction of such channels results in a number of disorders. For example, mutations identified in human and mouse calcium channel genes have been found to account for familial hemiplegic migraine, episodic ataxia type 2, cerebellar ataxia, absence epilepsy and seizures. Ophoff, et al., “Familial hemiplegic migraine and episodic ataxia type-2 are caused by mutations in the Ca2+gene CACNL1A4.” Cell (1996) 87, 543-552; Fletcher, et al., “Absence epilepsy in tottering mutant mice is associated with calcium channel defects.” Cell (1996) 87, 607-617; and Zhuchenko, et al., “Autosomal dominant cerebellar ataxia (SCA6) associated with small polyglutamine expansions in the α1A-voltage-dependent calcium channel.” Nature Genetics (1997) 15, 62-69.
Indeed, the clinical treatment of some disorders has been aided by the development of therapeutic calcium channel blockers. See, for example, Janis, et al., Calcium Channels: Their Properties, Functions, Regulation and Clinical Relevance (1991). CRC Press, London.
Calcium channels have been classified by their electrophysiological and pharmacological properties as T, L, N, P and Q types (for reviews see McCleskey, et al., Curr. Topics Membr. (1991) 39:295-326, and Dunlap, et al., Trends Neurosci. (1995) 18:89-98). T-type (or low voltage-activated) channels activate at relatively negative membrane potentials and are highly sensitive to changes in resting potential. The L, N and P/Q-type channels activate at more positive potentials and display diverse kinetics and voltage-dependent properties. There is some overlap in biophysical properties of the high voltage-activated channels, consequently pharmacological profiles are useful to distinguish them. L-type channels are sensitive to dihydropyridine (DHP) agonists and blockers, N-type channels are blocked by the Conus geographus peptide toxin, ω-conotoxin GVIA, and P-type channels are blocked by the peptide ω-agatoxin IVA from the venom of the funnel web spider, Agelenopsis aperta. The Q- and P-type channels appear very similar, and it has been suggested that they result from alternative splicing of a single gene (Bourinet, et al., “Phenotype variants of P- and Q-type calcium channels are generated by alternative splicing of the α1A subunit gene.” Nature Neuroscience (1999) 2:407-415.
The high voltage threshold calcium channels (L, N and P/Q) are complexes consisting of three distinct subunits (α1, α2δ and β) (reviewed by De Waard, et al., Ion Channels, Volume 4, (1997) edited by Narahashi, T. Plenum Press, New York). The α1 subunit is the major pore-forming subunit and contains the voltage sensor and binding sites for calcium channel modulators. The α2 subunit is mainly extracellular, and is disulfide-linked to the transmembrane δ subunit, both of which are derived from the same gene and are proteolytically cleaved in vivo. The β subunit is a non-glycosylated, hydrophilic protein with a high affinity of binding to a cytoplasmic region of the α1 subunit. A fourth subunit, γ, is unique to L-type Ca channels expressed in skeletal muscle T-tubules.
Molecular cloning has revealed the cDNA and corresponding amino acid sequences of six different types of α1 subunits corresponding to the high voltage threshold channels (α1A, α1B, α1C, α1D, α1E and α1S) and four types of β subunits (β1, β2, β3, and β4) (reviewed in Stea, A., Soong, T. W. and Snutch, T. P., “Voltage-gated calcium channels.” in Handbook of Receptors and Channels (1994), edited by R. A. North, CRC Press).
More recently, several α1 subunits corresponding to the low voltage gated T-type calcium ion channel have been cloned. Descriptions of these cloned α1 subunits may be found, for example, in PCT publications WO 98/38301 and WO 01/02561 as well as in U.S. Pat. Nos. 6,309,858 and 6,358,706, all incorporated herein by reference.
The α1 subunits are generally of the order of 2000 amino acids in length and contain 4 internal homologous repeats (domains I-IV) each having six putative alpha helical membrane spanning segments (S1-S6) with one segment (S4) having positively charged residues every third or fourth amino acid. There are a number of splice variant exceptions. Between domains II and III there is a cytoplasmic domain that is believed to mediate excitation-contraction coupling in α1S and which ranges from 100-400 amino acid residues among the subtypes. The domains I-IV make up roughly ⅔ of the molecule and the carboxy terminus adjacent to the S6 region of domain IV is believed to be on the intracellular side of the calcium channel. In the α1 subunits that code for the high voltage-gated channels, there is a consensus motif (QQ-E-L-GY-WI-E) downstream from the domain I S6 transmembrane segment that is a binding site for the β subunit. However, α1G, α1H and α1I, the only subunits thus far cloned coding for low voltage-gated channels, lack this binding site.
In some expression systems the high threshold α1 subunits alone can form functional calcium channels although their electrophysiological and pharmacological properties can be differentially modulated by coexpression with any of the four β subunits, and their efficiency is enhanced by the presence of α2. On the other hand, in general, the low voltage gated T-type channels generally function quite well when the α1 subunit is present alone. Perez-Reyes, et al., “Molecular characterization of a neuronal low-voltage-activated T-type calcium channel.” Nature (1998) 391: 896-900; Cribbs, et al., “Cloning and characterization of α1H from human heart, a member of the T-type Ca2+channel gene family.” Circ. Res. (1998) 83: 103-109 and McRory, et al., “Molecular and functional characterization of a family of rat brain T-type calcium channels.” J. Biol. Chem. (2001) 276: 3999-4011.
In the T-type α1 subunit, the pore region (P-region) in each of the four structural domains contains a diagnostic amino acid sequence implicated in channel permeability—i.e., the residues glutamate/glutamate/aspartate/aspartate (EEDD). This also distinguishes T-type channels from sodium (Na) channels where the P-region of the channels from the four domains contains the residues aspartate/glutamate/lysine/alanine (DEKA), and from high threshold calcium channels where the corresponding residues are glutamate/glutamate/glutamate/glutamate (EEEE).
The T-type channels are also distinct in that they do not possess an EF-hand calcium binding motif in the region carboxyl to domain IV S6, while all high threshold calcium channels contain a consensus sequence that is closely related to the EF-hand domain found in certain calcium binding proteins.
It is of considerable interest to identify compounds that modulate channel activity, for example, by blocking the flow of calcium and/or inhibiting the activation of calcium channels. One standard method to do so is through the use of patch clamp experiments. In these experiments, cells must be evaluated individually and in sequence by highly skilled operators, by measuring the calcium current across the cell membrane in response to changes of the membrane potential and/or application of test compounds. These experiments, while valid and informative, are very time consuming and not adaptable to high-throughput assays for compounds that modulate calcium ion channel activity.
For high-throughput assays of high voltage-gated calcium channel blockers, a more efficient assay is currently used which takes advantage of commercially available fluorophores that change their fluorescence emission in the presence of calcium ion. After loading cells expressing high voltage-gated calcium ion channels with such fluorophores, a single operator can measure calcium channel activity in hundreds of wells in parallel by exposing the cells to high levels of extracellular potassium ion. This simple technique is based on the observation that the resting potential of the cells is largely determined by the ratio of the extracellular versus the intracellular potassium ion concentrations. Normally, potassium is lower extracellularly than intracellularly, and produces a resting potential that is negative inside the cell. Increased levels of extracellular potassium, at concentrations close to that present intracellularly, will depolarize the membrane (abolishing the internal negativity), and activate calcium channels. Less activation of calcium channels will be observed if a blocker is applied to the cells.
It is known that calcium channels (and voltage-gated ion channels in general) can exist in three states: inactivated (not available for opening), resting (available for opening), and activated (open). Based on this pattern, in order for the calcium ion channels to respond to the potassium pulse, a substantial fraction of channels must be in the resting state, as opposed to the inactivated state. Typically, at the spontaneous resting membrane potential of −30 mV, about 40-70% of N-type calcium channels are in the resting state and available for opening. It is important to consider that transitions between each of these states is regulated by the membrane voltage. Moreover, the transition from inactivated state to resting state is slow, but the conversion of a resting to an activated channel, where the activated channel allows calcium ion influx, is quite fast. The return of the activated channels to the inactivated form is also relatively slow.
If a compound is successful in blocking calcium channel activation, calcium influx does not occur or occurs to a lesser extent and the fluorescence reading is lower or nonexistent, so this phenomenon can be used to identify modulators.
Attempts to perform this type of assay using low voltage-activated calcium channels (T-type) have not been successful in view of their inactivated status at the spontaneous membrane potential of −30 mV. At this potential, essentially all T-type channels are inactivated, and thus unavailable for activation by a high potassium pulse, or by any physiological stimulus. It has now been found that the fluorescence-based assay described above can be adapted to the T-type channel requirements by decreasing the membrane potential to about −70 mV before potassium ion activation, thus converting a sufficient number of T-type channels to the resting state.